Robert D. Stout

Macrophages Sequentially Change Their Functional Phenotype in Response to Changes in Microenvironmental Influences

Recent studies have described the development of distinct functional subsets of macrophages in association with cancer, autoimmune disease, and chronic infections. Based on the ability of Th1 vs Th2 cytokines to promote opposing activities in macrophages, it has been proposed that macrophages develop into either type 1 inflammatory or type 2 anti-inflammatory subsets. As an alternative to the concept of subset development, we propose that macrophages, in response to changes in their tissue environment, can reversibly and progressively change the pattern of functions that they express. As demonstrated herein, macrophages can reversibly shift their functional phenotype through a multitude of patterns in response to changes in cytokine environment. Macrophages display distinct functional patterns after treatment with IFN-γ, IL-12, IL-4, or IL-10 and additional functional patterns are displayed depending on whether the cytokine is present alone or with other cytokines and whether the cytokines are added before or concomitantly with the activating stimulus (LPS). Sequential treatment of macrophages with multiple cytokines results in a progression through multiple functional phenotypes. This ability to adapt to changing cytokine environments has significant in vivo relevance, as evidenced by the demonstration that macrophage functional phenotypes established in vivo in aged or tumor-bearing mice can be altered by changing their microenvironment. A concept of functional adaptivity is proposed that has important implications for therapeutic targeting of macrophages in chronic diseases that result in the dominance of particular functional phenotypes of macrophages that play a significant role in disease pathology.

Functional plasticity of macrophages: reversible adaptation to changing microenvironments

There has been substantial research activity in the past decade directed at phenotyping macrophage lineages and defining macrophage functional subsets or patterns of activity. The emphasis over the past 2–3 years has been to divide macrophage functional patterns into type 1 (Th1‐driven) or type 2 (Th2‐driven) functions. However, a huge array of environmental factors (including cytokines, chemokines, pattern recognition receptors, hormones) differentially regulates macrophage response patterns, resulting in the display of numerous distinct, functional phenotypes. Upon stimulation, a macrophage does not display just a single set of functions but rather displays a progression of functional changes in response to the progressive changes in its microenvironment. The remarkable ability of monocytes and tissue macrophages to adapt to changes in their microenvironment challenges the thesis that macrophages displaying unique tissue‐specific or response‐specific, functional patterns represent distinct lineages. With the exception of mature osteoclasts and mature dendritic cells, evidence supporting stable differentiation as the basis for macrophage functionl heterogeneity is equivocal. The concept of whether macrophages develop into functional subsets as opposed to continuously adapting their functional pattern in response to the changing environment of a progressive inflammatory response is important to resolve from the perspectives of therapeutic targeting and understanding the role of macrophages in disease pathogenesis.

Adenosine 5′-Monophosphate-Activated Protein Kinase Promotes Macrophage Polarization to an Anti-Inflammatory Functional Phenotype

Herein, we demonstrate a role of AMP-activated protein kinase (AMPK) as a potent counterregulator of inflammatory signaling pathways in macrophages. Stimulation of macrophages with anti-inflammatory cytokines (i.e., IL-10 and TGFβ) resulted in the rapid phosphorylation/activation of AMPK, whereas stimulation of macrophages with a proinflammatory stimulus (LPS) resulted in AMPK dephosphorylation/inactivation. Inhibition of AMPKα expression by RNA interference dramatically increased the mRNA levels of LPS-induced TNF-α, IL-6, and cyclooxygenase-2. Likewise, expression of a dominant negative AMPKα1 in macrophages enhanced TNF-α and IL-6 protein synthesis in response to LPS stimulation, while diminishing the production of IL-10. In contrast, transfection of macrophages with a constitutively active form of AMPKα1 resulted in decreased LPS-induced TNF-α and IL-6 production, and heightened production of IL-10. In addition, we found that AMPK negatively regulated LPS-induced IκB-α degradation and positively regulated Akt activation, accompanied by inhibition of glycogen synthase kinase β and activation of CREB. Thus, AMPK directs signaling pathways in macrophages in a manner that suppresses proinflammatory responses and promotes macrophage polarization to an anti-inflammatory functional phenotype.

Immunosenescence and macrophage functional plasticity: dysregulation of macrophage function by age-associated microenvironmental changes

Summary:  The macrophage lineage displays extreme functional and phenotypic heterogeneity, which appears to be because, in large part, of the ability of macrophages to functionally adapt to changes in their tissue microenvironment. This functional plasticity of macrophages plays a critical role in their ability to respond to tissue damage and/or infection and to contribute to clearance of damaged tissue and invading microorganisms, to recruitment of the adaptive immune system, and to resolution of the wound and of the immune response. Evidence has accumulated that environmental influences, such as stromal function and imbalances in hormones and cytokines, contribute significantly to the dysfunction of the adaptive immune system. The innate immune system also appears to be dysfunctional in aged animals and humans. In this review, the hypothesis is presented and discussed that the observed age‐associated ‘dysfunction’ of macrophages is the result of their functional adaptation to the age‐associated changes in tissue environments. The resultant loss of orchestration of the manifold functional capabilities of macrophages would undermine the efficacy of both the innate and adaptive immune systems. The macrophages appear to maintain functional plasticity during this dysregulation, making them a prime target of cytokine therapy that could enhance both innate and adaptive immune systems.

IL-12 Rapidly Alters the Functional Profile of Tumor-Associated and Tumor-Infiltrating Macrophages In Vitro and In Vivo

Tumor-associated macrophages (TAMs) play a major role in promoting tumor growth and metastasis and in suppressing the antitumor immune response. Despite the immunosuppressive environment created by the tumor and enforced by tumor-associated macrophages, treatment of tumor-bearing mice with IL-12 induces tumor regression associated with appearance of activated NK cells and activated tumor-specific CTLs. We therefore tested the hypothesis that IL-12 treatment could alter the function of these tumor-associated suppressor macrophages. Analysis of tumor-infiltrating macrophages and distal TAMs revealed that IL-12, both in vivo and in vitro, induced a rapid (<90 min) reduction of tumor supportive macrophage activities (IL-10, MCP-1, migration inhibitory factor, and TGFβ production) and a concomitant increase in proinflammatory and proimmunogenic activities (TNF-α, IL-15, and IL-18 production). Similar shifts in functional phenotype were induced by IL-12 in tumor-infiltrating macrophages isolated from the primary tumor mass and in TAMs isolated from lung containing metastases, spleen, and peritoneal cavity. Therefore, although TAMs display a strongly polarized immunosuppressive functional profile, they retain the ability to change their functional profile to proinflammatory activities given the appropriate stimulus. The ability of IL-12 to initiate this functional conversion may contribute to early amplification of the subsequent destructive antitumor immune response.

Functional plasticity of macrophages: in situ reprogramming of tumor-associated macrophages

The extent to which the functional heterogeneity of Mφs is dependent on the differentiation of functional sublineages remains unresolved. One alternative hypothesis proposes that Mφs are functionally plastic cells, which are capable of altering their functional activities progressively in response to progressively changing signaling molecules generated in their microenvironment. This “functional plasticity” hypothesis predicts that the functionally polarized Mφs in chronic pathologies do not represent Mφ sublineages but rather, are mutable phenotypes sustained by chronic signaling from the pathological environment. Solid TAMφs are chronically polarized to provide activities that support tumor growth and metastasis and suppress adaptive immune responses. In support of the functional plasticity hypothesis, administration of slow‐release microsphere‐encapsulated IL‐12 successfully reprogrammed TAMφs in situ, reducing Mφ support of tumor growth and metastasis and enhancing Mφ proimmunogenic activities. Increased knowledge of how Mφ function is regulated and how polarized Mφs can be reprogrammed in situ will increase our ability to control Mφ function in a variety of pathological states, including cancer and chronic inflammatory disease.

CD40 Signaling of Monocyte Inflammatory Cytokine Synthesis through an ERK1/2-dependent Pathway A TARGET OF INTERLEUKIN (IL)-4 AND IL-10 ANTI-INFLAMMATORY ACTION

Ligation of CD40 on monocytes through its interaction with CD40 ligand (CD154) present on activated T helper cells, results in activation of monocyte inflammatory cytokine synthesis and rescue of monocytes from apoptosis induced through serum deprivation. Both of these consequences of CD40 stimulation have been shown to be dependent on the induction of protein tyrosine kinase activity. CD40-mediated activation of protein tyrosine kinase activity and subsequent inflammatory cytokine production are abrogated by treatment of monocytes with the T helper type 2 cytokines interleukin 4 (IL-4) and interleukin 10 (IL-10). In the current study we demonstrate that stimulation of monocytes through CD40 resulted in the phosphorylation and activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein kinases, whereas phosphorylation of mitogen-activated protein kinases family members p38 and c-Jun N-terminal kinase was not observed in response to this stimuli over the time course examined. PD98059, an inhibitor of the upstream activator of ERK1/2, the MAP/ERK kinase MEK1/2, suppressed IL-1β and tumor necrosis factor-α production in a dose-dependent fashion. Pretreatment of monocytes with IL-4 and IL-10 inhibited CD40-mediated activation of ERK1/2 kinase activity when used individually, and are enhanced in effectiveness when used in combination. Together, the data demonstrate that CD40-mediated induction of IL-1β and tumor necrosis factor-α synthesis is dependent on a MEK/ERK pathway which is obstructed by signals generated through the action of IL-4 and IL-10.

Macrophage CD40 signaling: A pivotal regulator of disease protection and pathogenesis

Macrophages reside in all tissues as resident populations and as immigrants recruited in response to tissue injury, inflammation or pathogen invasion. Under normal conditions, macrophages contribute to tissue homeostasis and provide innate immune surveillance. Both macrophages and their progenitors, bone marrow-derived monocytes, constitutively express the tumor necrosis factor receptor superfamily member, CD40, and are capable of a robust response to CD40 ligation resulting in the induction or enhancement of expression of genes with a predominantly pro-inflammatory function. CD40 signaling in macrophages in the context of host responses to pathogens plays a crucial role in host defense. However, macrophage responses to CD40 ligation in the context of autoimmune and cardiovascular disease contribute to disease pathogenesis. In this review, we discuss the role of CD40 in both protective and destructive processes, including the signaling pathways engaged and the factors capable of modulating CD40 signal transduction.

Regulation of Th17 Differentiation by Epidermal Fatty Acid-Binding Protein

Epidermal fatty acid-binding protein, E-FABP, a lipid chaperone, has been shown to regulate the inflammatory function of macrophages and dendritic cells. Herein, we demonstrate that T cell expression of E-FABP promotes Th17 differentiation, while counterregulating development of FoxP3+ regulatory T cells (Tregs). In response to immunization with myelin oligodendrocyte glycoprotein peptide (MOG35–55), E-FABP-deficient mice generated reduced levels of Th17 cells and elevated levels of Tregs, as compared with wild-type mice. Likewise, naive CD4+ T cells isolated from E-FABP-deficient mice showed reduced expression of IL-17 and enhanced expression of FoxP3, in vitro, when subjected to Th17 or Treg polarizing conditions, respectively. It has been demonstrated previously that IL-21, induced by IL-6, stimulates the expression of the nuclear receptors retinoic acid-related orphan receptor (ROR)γt and RORα, which in turn induce expression of IL-17. We found that the impaired Th17 differentiation by E-FABP-deficient CD4+ T cells was associated with lower levels of IL-21 expression in response to IL-6, as well as reduced expression of RORγt and RORα. However, E-FABP-deficient CD4+ T cells expressed significantly higher levels of the nuclear receptor peroxisome proliferator-activating receptor (PPAR)γ than did wild-type CD4+ T cells, and treatment with the PPARγ antagonist GW9662 restored expression of IL-21, RORγt, RORα, and IL-17 by E-FABP-deficient T cells to wild-type levels. The negative influence of E-FABP deficiency on IL-17 expression was attributed to PPARγ-mediated suppression of IL-6-induced STAT3 activity. Thus, taken together, our data indicate that expression of E-FABP by CD4+ T cells contributes to the control of IL-6 stimulation of the IL-21/ROR/IL-17 pathway and to the Th17/Treg counterbalance.

Deficiency of Fatty Acid-Binding Proteins in Mice Confers Protection from Development of Experimental Autoimmune Encephalomyelitis

Fatty acid-binding proteins (FABPs) act as intracellular receptors for a variety of hydrophobic compounds, enabling their diffusion within the cytoplasmic compartment. Recent studies have demonstrated the ability of FABPs to simultaneously regulate metabolic and inflammatory pathways. We investigated the role of adipocyte FABP and epithelial FABP in the development of experimental autoimmune encephalomyelitis to test the hypothesis that these FABPs impact adaptive immune responses and contribute to the pathogenesis of autoimmune disease. FABP-deficient mice exhibited a lower incidence of disease, reduced clinical symptoms of experimental autoimmune encephalomyelitis and dramatically lower levels of proinflammatory cytokine mRNA expression in CNS tissue as compared with wild-type mice. In vitro Ag recall responses of myelin oligodendrocyte glycoprotein 35–55-immunized FABP−/− mice showed reduced proliferation and impaired IFN-γ production. Dendritic cells deficient for FABPs were found to be poor producers of proinflammatory cytokines and Ag presentation by FABP−/− dendritic cells did not promote proinflammatory T cell responses. This study reveals that metabolic-inflammatory pathway cross-regulation by FABPs contributes to adaptive immune responses and subsequent autoimmune inflammation.